# Spectroscopy Investigations of Metalloenzyme Mechanisms

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2022 · $392,500

## Abstract

Enzymes using metal centers and/or organic radicals play many crucial roles in the fundamental
biochemistry of human health, with deficiencies in their bioassembly or enzymatic functions
associated with various diseases. The R. David Britt laboratory is using advanced spectroscopic
techniques such as multifrequency electron paramagnetic resonance (EPR) to understand the
assembly and catalytic mechanism of a number of such metal and radical centers. Many
important enzymes involved in multielectron oxidation or reduction reactions employ metal
clusters in their catalysis. The Britt laboratory is studying how such clusters are assembled by
identifying and interrogating assembly intermediates with their spectroscopic methods. For
example the [Fe-Fe] hydrogenase enzyme uses a complex multinuclear Fe-S “H-cluster”,
containing organometallic Fe-CO and Fe-CN components, to catalyze reversible interconversion
of H2 with protons and electrons. How does nature safely assemble such a center involving
potentially dangerous CO and CN- species? The Britt laboratory is exploring how a radical reaction
catalyzed by HydG, a member of the “radical SAM” superfamily of enzymes, safely forms
Fe(CO)x(CN)y organometallic synthons at the earliest stage of cluster synthesis and how these
are processed by other maturase enzymes in the synthesis of the catalytic cluster. Magnetic
nuclear isotopes (e.g. 13C, 15N, 57Fe) provide important magnetic interactions with paramagnetic
forms of such clusters, as observed by EPR-based methods such as electron-nuclear double
resonance (ENDOR). These and other nuclei provide handles for other spectroscopic probes of
FeS cluster assembly such as Mössbauer, NMR, and FTIR. Their ability to assemble the active
site enzymatically via cell-free synthesis allows the Britt laboratory to isotope-edit the assembled
H-cluster and probe intermediates in hydrogen catalysis with isotope specific spectroscopies.
Parallel experiments are unraveling the biosynthesis of the complex Fe-S “M-cluster” at the heart
of the nitrogenase enzyme, which can incorporate Mo or V or an additional Fe in its active site.
The Britt lab is studying other members of the radical SAM superfamily that can carry out a wide
variety of reactions such as organic cofactor and vitamin biosynthesis and post translational
modifications of short peptides. Further investigations with EPR and other spectroscopies will
probe metalloenzymes and radical enzymes with diverse functions, including NO and Ca2+
signaling, organohalide detoxification, metal ion sequestration and homeostasis, and substrate
oxygen insertion. In such projects the Britt laboratory works closely with a wide variety of
collaborators working on numerous NIH-supported research projects.

## Key facts

- **NIH application ID:** 10378679
- **Project number:** 5R35GM126961-05
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** R. David Britt
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $392,500
- **Award type:** 5
- **Project period:** 2018-04-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10378679

## Citation

> US National Institutes of Health, RePORTER application 10378679, Spectroscopy Investigations of Metalloenzyme Mechanisms (5R35GM126961-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10378679. Licensed CC0.

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